Contribution of electrostatics to the binding of pancreatic-type ribonucleases to membranes.

Medical Scientist Training Program and Graduate Program in Biophysics, ‡Department of Biochemistry, and §Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.

Abstract

Pancreatic-type ribonucleases show clinical promise as chemotherapeutic agents but are limited in efficacy by the inefficiency of their uptake by human cells. Cellular uptake can be increased by the addition of positive charges to the surface of ribonucleases, either by site-directed mutagenesis or by chemical modification. This observation has led to the hypothesis that ribonuclease uptake by cells depends on electrostatics. Here, we use a combination of experimental and computational methods to ascertain the contribution of electrostatics to the cellular uptake of ribonucleases. We focus on three homologous ribonucleases: Onconase (frog), ribonuclease A (cow), and ribonuclease 1 (human). Our results support the hypothesis that electrostatics are necessary for the cellular uptake of Onconase. In contrast, specific interactions with cell-surface components likely contribute more to the cellular uptake of ribonuclease A and ribonuclease 1 than do electrostatics. These findings provide insight for the design of new cytotoxic ribonucleases.

Computational calculation of the binding of RNase 1 to model membranes. (A) Poisson–Boltzmann calculations of the electrostatic free energy of interaction between RNase 1 (PDB entry 1z7x), rotated using Euler angles (θ= 0–360°; ψ= 0–180°; increments of 15°), with a model membrane with an anionic fraction of 1 electron per 130 Å2. (B) A coarse-grained representation of trajectories from a 2-ns IMM1–GC simulation that result in a protein·membrane complex. Each point represents a snapshot from the trajectory. (C) Depiction of RNase 1 with a model membrane in the most energetically favorable orientation from PB calculations. In the images of Figures 3–, the N-terminal helix is shown in the center-rear; and Arg and Lys (blue), and Asp and Glu (red) side chains are shown explicitly. (D) Depiction of RNase 1 with model membrane in the least energetically favorable orientation from PB calculations.

Computational calculation of the binding of RNase A to model membranes. (A) Poisson–Boltzmann calculations of the electrostatic free energy of interaction between RNase A (PDB entry 1kf5), rotated using Euler angles (θ= 0–360°; ψ= 0–180°; increments of 15°), with a model membrane with an anionic fraction of 1electron per 130 Å 2. (B) A coarse-grained representation of trajectories from a 2-ns IMM1–GC simulation that result in a protein·membrane complex. (C) Depiction of RNase A with a model membrane in the most energetically favorable orientation from PB calculations. (D) Depiction of RNase A with a model membrane in the least energetically favorable orientation from PB calculations.

Computational calculation of ONC binding to model membranes. (A) Poisson–Boltzmann calculations of the electrostatic free energy of interaction between ONC (PDB entry entry 1onc), rotated using Euler angles (θ= 0–360°; ψ= 0–180°; increments of 15°), with a model membrane with an anionic fraction of 1electron per 130 Å 2. (B) A coarse-grained representation of trajectories from a 2-ns IMM1–GC simulation that result in a protein·membrane complex. (C) Depiction of ONC with a model membrane in the most energetically favorable orientation from PB calculations. (D) Depiction of ONC with a model membrane in the least energetically favorable orientation from PB calculations. (E) A coarse-grained representation of trajectories from a 2-ns IMM1–GC simulation that do not result in a protein·membrane complex.